Exposure to ionising radiation is known to cause deleterious effects in humans, many of which are thought to be mediated by the induction of somatic and germline mutation. A new type of assay with which to determine levels of mutation directly from human DNA samples has recently been developed by the author. The assay uses the Polymerase Chain reaction (PCR), an enzymatic technique for amplifying genetic sequences to detect deletion mutations of the mitochondrial genome which occur in human DNA. Basal (spontaneous) levels of a particular deletion mutation (delta1) were detected somatic tissues of normal adult humans with this assay, and appear to increase with age (Cortopassi and Amheim, 1990). The identical,delta1 deletion occurs at high level in some rare, sporadically inherited human genetic diseases. A PCR-based assay may have several intrinsic advantages over other existing assays of radiation-induced somatic mutation. The number of mutant molecules can be directly determined from purified DNA; therefore, the level of somatic mutation can be determined from any source from which DNA is available, including tissue culture or fresh, frozen, or formalin-fixed human mitotic or postmitotic tissues. Because the assay is DNA-based, no "expression time" subsequent to occurrence of the mutation is required for detection. The high copy number of mitochondrial DNA (mtDNA) and deficiency in recombination and repair of mtDNA deletions might make this system an optimal genetic marker of radiation exposure, since the effective target size is relatively large, and multiple hits in mitochondrial targets are known not to be lethal to the cell. Aims of this proposal include the development of a PCR-based assay of a nuclear gene (ADA) which is thought to contain a "deletion hotspot", determination of the effect of ionising radiation on absolute levels of mtDNA and ADA deletions in human tissue culture, and its effect on relative frequencies of particular mtDNA deletions (the mutation spectrum) in tissue culture. Preliminary data which bear on several of these objectives have been collected. The experimental design of the radiation experiments involves exposure of cells to the desired radiation dose, preparation of DNA from the exposed cells, and quantitative determination of the number of deleted forms relative to normal mtDNA or ADA genes using techniques that have been developed (Cortopassi and Amheim, 1990).